Summary

The septins are a family of proteins involved in cytokinesis and other
aspects of cell-cortex organization. In a two-hybrid screen designed to
identify septin-interacting proteins in Drosophila, we isolated
several genes, including homologues (Dmuba2 and Dmubc9) of
yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in
the activation and conjugation, respectively, of the ubiquitin-like protein
Smt3p/SUMO, which becomes conjugated to a variety of proteins through this
pathway. Uba2p functions together with a second protein, Aos1p. We also cloned
and characterized the Drosophila homologues of AOS1
(Dmaos1) and SMT3 (Dmsmt3). Our biochemical data
suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating
enzymes for DmSmt3, implying that this protein-conjugation pathway is well
conserved in Drosophila. Immunofluorescence studies showed that
DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial
blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in
the nuclei during interphase but dispersed throughout the cells during
mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These
data suggest that DmSmt3 could modify target proteins both inside and outside
the nuclei. We did not observe any concentration of DmUba2 at sites where the
septins are concentrated, and we could not detect DmSmt3 modification of the
three Drosophila septins tested. However, we did observe DmSmt3
localization to the midbody during cytokinesis both in tissue-culture cells
and in embryonic mitotic domains, suggesting that DmSmt3 modification of
septins and/or other midzone proteins occurs during cytokinesis in
Drosophila.

The functions of modification by Smt3/SUMO and other ubiquitin-like
proteins are not entirely clear, but they appear to include regulation of
protein localization (RanGAP1), transcriptional activity (p53 and c-Jun), and
protein stability (Mdm2). Unlike ubiquitin itself
(
Hochstrasser, 1996), other
ubiquitin-like proteins do not appear to target modified proteins for
degradation and in fact may stabilize target proteins by preventing their
ubiquitination (
Hochstrasser,
2000;
Melchior,
2000).

The ubiquitin-like proteins have differing and generally low levels of
sequence similarity to ubiquitin itself; for example, yeast Smt3p and
ubiquitin have only 17% identity in their amino acid sequences. Nonetheless,
the respective conjugation pathways have many common features
(
Hochstrasser, 2000;
Melchior, 2000). In most
cases, ultimate conjugation is by an isopeptide bond between a C-terminal
glycine on ubiquitin or the ubiquitin-like protein and the ϵ-amino group
of a lysine in the target protein. The conjugation reaction involves an `E2'
conjugating enzyme and, at least in some cases, an `E3' protein ligase
involved in target-protein recognition. The conjugating enzyme receives the
ubiquitin or ubiquitin-like protein from an `E1' activating enzyme, which
first adenylates the C-terminus of ubiquitin or the ubiquitin-like protein and
then links it by a thiolester bond to a cysteine in the E1 enzyme. The
enzymatic mechanisms of the various E1 enzymes are similar, and the enzymes
themselves are structurally related; this is also true of the various E2
enzymes. In the case of yeast Smt3p, activation is carried out by a
heterodimer of Uba2p and a second protein, Aos1p, and links the C-terminus of
Smt3p to the active site cysteine (Cys 177) in Uba2p
(
Johnson et al., 1997). Smt3p
is then transferred and conjugated by another thiolester bond to Cys 93 in
Ubc9p (
Johnson and Blobel,
1997;
Schwarz et al.,
1998). The available data suggest that the same enzymatic
machinery is used for SUMO-1 conjugation in mammalian cells
(
Desterro et al., 1997;
Desterro et al., 1999;
Hochstrasser, 2000;
Melchior, 2000;
Okuma et al., 1999). Although
E3 enzymes have not yet been identified for most ubiquitin-like proteins
(
Hochstrasser, 2000;
Melchior, 2000), recent
studies have identified two possible E3 enzymes for Smt3/SUMO modification
(
Johnson and Gupta, 2001;
Kahyo et al., 2001;
Takahashi et al., 2001).

In this study, we sought septin-interacting proteins in Drosophila
and attempted to elucidate the relationship between the septins and the Smt3
conjugation system in this organism. In addition, we used biochemical and cell
biological approaches to characterize the Drosophila Smt3 conjugation
pathway and investigate its possible functions.

Two-hybrid assays and screening

Two-hybrid assays were performed as described previously
(
DeMarini et al., 1997;
Fields and Sternglanz, 1994;
Gyuris et al., 1993) using the
LexA DNA-binding domain (DBD) plasmid pEG202
(
Ausubel et al., 1994) and
activation domain (AD) plasmid pJG4-5
(
Ausubel et al., 1994) or
pJG4-5PL (
DeMarini et al.,
1997). The baits used for screening contained full-length
sequences of pnut, sep1, and sep2 (J. C. Adam et al.,
unpublished). The libraries used (provided by R. Finley, Harvard Medical
School, Boston, MA) were RFLY1, a Drosophila embryonic cDNA library,
and RFLY5, an imaginal-disc cDNA library, both in pJG4-5. Each bait plasmid
was co-transformed into yeast strain EGY48R with each of the two libraries.
Transformants were plated onto Synthetic Minimal plates
(
Guthrie and Fink, 1991)
containing 2% galactose + 1% raffinose and grown for 3-6 days to select
Leu+ clones, which were further evaluated using both filter and
liquid-culture β-galactosidase assays
(
Ausubel et al., 1994).
Plasmids from positive clones were rescued into E. coli strain JMB9
and retested by cotransformation into EGY48R together with one of the
pEG202-based plasmids and measurement of β-galactosidase activity.
Inserts from positive plasmids were then sequenced. We screened>
106 transformants for each library with each bait.

Additional two-hybrid tests used the following plasmids. pJG4-5PL,
expressing full-length anillin, was provided by Julie Brill (Hospital for Sick
Children, Toronto, Canada). Construction of plasmids expressing N- or
C-terminal fragments of Pnut (Pnut-N, amino acids 1-426; Pnut-C, amino acids
407-539), Sep1 (Sep1-N, amino acids 1-325; Sep1-C, amino acids 306-361), or
Sep2 (Sep2-N, amino acids 1-337; Sep2-C, amino acids 318-419) in pEG202 will
be described in detail elsewhere (J. C. Adam et al., unpublished). Full-length
Dmuba2 and Dmaos1 and the 5′ (amino acids 1-350) and
3′ (amino acids 351-700) halves of Dmuba2 were amplified by PCR
using the cloned genes (see below) as templates and the primers shown in
Table 1. The amplified
Dmuba2 and its fragments were cloned into pEG202 and pJG4-5PL at
their EcoRI sites, and Dmaos1 was cloned into pEG202 and
pJG4-5PL at their XhoI sites. The full-length genes and the
Dmuba2 C-terminal constructs contain the original stop codons,
whereas the Dmuba2 N-terminal constructs use a stop codon in the
vector downstream of the polylinker.

Isolation of full-length Dmuba2, Dmaos1, and Dmsmt3
clones

Two independent positive clones from the two-hybrid screen encoded
C-terminal fragments (including the putative stop codon) of the same gene. To
isolate the 5′ end of this gene, we identified an EST clone (LD03967)
from the Berkeley Drosophila Genome Project (BDGP) that extended
further in the 5′ direction and then performed PCR as described
previously (
McCurdy and Kim,
1998), using a Schneider cell cDNA library in vector pDB20
(
Becker et al., 1991) as
template and primers (
Table 1)
that corresponded to vector sequences and to the 5′ end of the LD03967
sequences, respectively. In aggregate, the cDNA sequences revealed an
apparently complete ORF of 2,100 bp that had similarity to yeast UBA2
(see Results). Full-length Dmuba2 was then cloned by PCR using the
primers shown in
Table 1, the
Schneider cell cDNA library as the template, and plasmid pGEM-T (Promega). We
mapped Dmuba2 to position 66B6-66B10 on chromosome arm 3L using a
high-density filter of P1 clones (Genome Systems, St. Louis, MO), as
subsequently confirmed by data from the genome project (see FlyBase).

Full-length clones of Dmaos1 and Dmsmt3 were obtained by
identifying and sequencing BDGP EST clones (GM10027 and GM01812) that
contained apparently complete ORFs with similarity to yeast Aos1p and Smt3p,
respectively (see Results).

To prepare DmUba2-specific antibodies, DNA encoding DmUba2 amino acids
553-700 was amplified by PCR using one of the two-hybrid clones as the
template and the primers shown in
Table
1. The products were cut with EcoRI and cloned into
pGEX-1 (Amersham-Pharmacia), producing pGEX-DmUba2, and into pMAL-c2 (New
England Biolabs, Beverly, MA), producing pMAL-DmUba2. The resulting
glutathione-S-transferase (GST)- and maltose-binding protein
(MBP)-fusion proteins were expressed, purified, and used to raise antibodies
in rabbits by standard methods (
Ausubel et
al., 1994) (Cocalico Biologicals, Reamstown, PA). After five
boosts, antibodies raised against GST-DmUba2 were affinity purified using
MBP-DmUba2 that had been subjected to SDS-PAGE and blotted to nitrocellulose
(
Pringle et al., 1989).

To prepare DmSmt3-specific antibodies, a His6-tagged DmSmt3
protein was generated. Dmsmt3 codons 1-88 were amplified by PCR using
Canton S genomic DNA (
Sullivan et al.,
2000) as the template and the primers shown in
Table 1. The resulting product
and plasmid pQE30 (Qiagen) were digested with BamHI and
HindIII and ligated together to produce pQE30-DmSmt3, which encodes
DmSmt3 tagged at its N-terminus with His6 and truncated after the
two glycines that presumably represent the C-terminus of the mature endogenous
protein (
Johnson et al., 1997;
Kamitani et al., 1997).
His6DmSmt3 was expressed in strain JM109 and purified on Ni-NTA
columns as recommended by Qiagen, and rabbit antibodies were raised using
standard protocols (Cocalico Biologicals). After four boosts, DmSmt3-specific
antibodies were affinity purified using His6DmSmt3 that had been
subjected to SDS-PAGE and blotted to nitrocellulose (see above).

To express Dmuba2 in yeast under GAL-promoter control, we
amplified full-length Dmuba2 by PCR using the cloned cDNA as template
and the primers shown in
Table
1, cut the product with SalI, and cloned it into YCpIF2
(
Foreman and Davis, 1994),
producing YCpIF2-DmUba2. Strain JD90-1A [pIS2-ts10] (see above) was
transformed with YCpIF2-DmUba2 and grown under conditions selective for both
plasmids and inducing or noninducing for the GAL promoter. Yeast
extracts were then prepared as described previously
(
Ausubel et al., 1994)

To prepare fly extracts for immunoprecipitation (IP), 0-16-hour-old embryos
were collected, dechorionated, and rinsed as described previously
(
Pai et al., 1996). 100 μl
of embryos were homogenized at 0°C in extraction buffer (500 μl of CER
I plus 27.5 μl of CER II; both reagents from the NE-PER kit, Pierce,
Rockford, IL). The extract was added to 1 ml of NET buffer containing
phosphatase and protease inhibitors (
Pai
et al., 1996) and centrifuged for 10 minutes at 15,000
g in a microfuge at 4°C, and the supernatant was
collected. IPs were carried out as described previously
(
Peifer, 1993) using purified
anti-DmUba2 (at 1:75), anti-DmUbc9 (at 1:200), or monoclonal anti-myc 9E10 (at
1:20). IPs were analyzed by SDS-PAGE and immunoblotting.

In vitro protein-binding assays

Codons 1-76 of a Drosophila ubiquitin gene
(
Lee et al., 1988) were
amplified by PCR using the Schneider cell cDNA library as the template and the
primers shown in
Table 1. The
resulting fragment was then cloned into pQE30 using KpnI and
HindIII. The resulting plasmid encodes ubiquitin (DmUb) tagged at its
N-terminus with His6 and truncated after the Gly-Gly that
presumably represents the C-terminus of the mature endogenous protein.
His6-DmSmt3 (see above) and His6-DmUb were expressed
separately in strain JM109 as recommended by Qiagen and purified using the
B-PER kit (Pierce). Fly embryo extracts were prepared as described above
except that homogenization was in 1 ml of RIPA buffer (50 mM Tris-HCl, pH 8.5,
300 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) containing 20 mM
imidazole, 2 mM ATP, 5 mM MgCl2, 0.1 mM DTT, 50 mM NaF, and
protease inhibitors (see above). Extract was centrifuged for 10 minutes at
8000 g in a microfuge at 4°C, and 5 μg of
His6-DmSmt3 or His6-DmUb was added to 450 μl of
supernatant. After incubation at 23°C for 15 minutes, 15 μl of Ni-NTA
beads were added, and the mixture was incubated at 4°C for 45 minutes. The
beads were washed four times in RIPA containing 20 mM imidazole, 50 mM NaF,
and protease inhibitors (as above). For analysis under reducing conditions,
beads were suspended in standard 1× Laemmli buffer and analyzed by
immunoblotting as described above. For analysis under nonreducing conditions,
beads were suspended in 1× Laemmli buffer without β-mercaptoethanol
and heated at 68°C for 10 minutes prior to SDS-PAGE and
immunoblotting.

Immunofluorescence microscopy

Embryos collected after development at 25°C were washed and
dechorionated as described above, then fixed, devitellinized, and stained as
described (
Cox et al., 1996).
The ages of syncytial-blastoderm embryos were determined by counting the
nuclei in longitudinal sections. Standard morphological criteria
(
Roberts, 1986) were used to
identify other developmental stages. Clone-8 cells were washed three times
with PBS, fixed with 2% paraformaldehyde in PBS for 10-15 minutes at 23°C,
washed twice with PBS, treated with primary antibodies in Solution H (PBS
containing 0.1% Triton X-100 and 1% normal goat serum) for 1 hour at 23°C,
washed once with PBS, treated with secondary antibodies in Solution H for 1
hour at 23°C, and washed twice again with PBS. The primary antibodies used
were purified anti-DmUba2 (at 1:50) and anti-DmSmt3 (at 1:75), monoclonal
anti-Pnut antibody 4C9 (at 1:3), and anti-β-tubulin (at 1:100). FITC- and
rhodamine-conjugated goat anti-rabbit-IgG and goat anti-mouse-IgG (Jackson
ImmunoResearch, West Grove, PA) were used at 1:500; Alexa Fluor 488-conjugated
goat anti-rabbit-IgG (Molecular Probes, Eugene, OR) and Cy3-conjugated goat
anti-mouse-IgG (Jackson ImmunoResearch) were used at 1:1000. Embryos and cells
were mounted in AquaPolymount (Polysciences, Warrington, PA), then observed
and photographed using a Zeiss LSM 410 confocal microscope.

Results

Identification of genes by two-hybrid interactions with
Drosophila septins

To identify proteins interacting with the Drosophila septins, we
conducted two-hybrid screens using Pnut, Sep1, and Sep2 as baits (see
Materials and Methods). These screens identified 27 positive clones that
proved to represent eight genes (
Table
2). Among these were the other septins, as expected from other
data indicating that septins interact with one another (see Discussion). In
addition, the screens identified Drosophila homologues
(Dmuba2 and Dmubc9) of yeast UBA2 and
UBC9, whose products are involved in the activation and conjugation
of the ubiquitin-like molecule Smt3p/SUMO (see Introduction). These screening
results and the recent discovery of Smt3p modification of septins in S.
cerevisiae (
Johnson and Blobel,
1999;
Takahashi et al.,
1999) (P. Meluh, personal communication) stimulated us to study
the Smt3p/SUMO conjugation machinery in Drosophila.

Two-hybrid interactions of Drosophila septins with each other,
sumoylation enzymes, and other
proteins
a

In further two-hybrid analyses, the C-terminal portion of DmUba2 interacted
strongly with full-length Sep1 and Sep2 and with the N-terminal portion of
Sep1. Interactions were also detected with the N-terminal portions of Pnut and
Sep2 and with the C-terminal portions of Sep1 and Sep2
(
Table 2). In contrast, a
full-length AD-DmUba2 fusion showed none of these interactions
(
Table 2), although other
studies (see
Table 3) indicated
that this fusion was functional for other interactions. Interestingly,
full-length AD-DmUbc9 showed a pattern of interactions very similar to those
seen with the C-terminal portion of DmUba2.

The other genes identified in the screens had not been described
previously; we designated them sip1-sip4 (for septin-interacting
protein). sip1 (Accession No. AF221101; Drosophila genome
annotation No. CG7238) encodes a protein with predicted P-loop and coiled-coil
domains; it appeared to interact specifically with the C-terminal portion of
Pnut (
Table 2).
sip2-sip4 encode proteins without obviously informative motifs.
sip2 (CG9188) encodes a protein that interacted with full-length Sep1
and Sep2 but not with Pnut. sip3 (CG1937) encodes a protein that
appeared to interact specifically with the C-terminal portion of Sep2.
sip4 was identified independently as dip2 (Dorsal
interacting protein 2) (
Bhaskar et al.,
2000); it encodes a protein that interacted with all of the Sep1
and Sep2 fusions and (weakly) with the N-terminal portion of Pnut
(
Table 2).

Cloning and sequence analysis of Dmuba2 and
Dmubc9

We obtained a full-length clone of Dmuba2 as described in the
Materials and Methods. Sequencing (Accession No. AF193553) showed that the
predicted DmUba2 contains 700 amino acids and has 29% sequence identity to
yeast Uba2p and 48% identity to human hUba2, as observed also by others
(
Bhaskar et al., 2000;
Long and Griffith, 2000;
Donaghue et al., 2000). Like its homologues and other E1-type enzymes, DmUba2
contains an ATP-binding motif (amino acids 26-31) and the consensus Cys (C175)
corresponding to those essential for thiolester bond formation in other
E1-type enzymes (
Desterro et al.,
1999;
Dohmen et al.,
1995). Our original two-hybrid clone of Dmubc9 appeared
to be full length by comparison to yeast UBC9 and our sequence for
Dmubc9 agreed with that reported by Joanisse et al.
(
Joanisse et al., 1998).

We then raised antibodies to DmUba2 (see Materials and Methods). The
affinity-purified antibodies recognized mainly one polypeptide of apparent
molecular weight ∼97 kDa (
Fig.
1A), which is presumably DmUba2 (predicted molecular weight, 77.5
kDa). Support for this conclusion was obtained by expressing Dmuba2
under GAL-promoter control in yeast. When cells were grown under
inducing conditions, the antibodies recognized primarily a polypeptide of
apparent molecular weight ∼97 kDa (
Fig.
1B, lane 1) that was absent when cells were grown under repressing
conditions (
Fig. 1B, lane 2).
Similarly anomalous low mobility on SDS-PAGE has been noted for both Uba2p and
hUba2 (
Desterro et al., 1999;
Dohmen et al., 1995).

Identification of DmAos1 and its interaction with DmUba2

In S. cerevisiae, the E1 enzyme for ubiquitin activation is the
1024 amino-acid Uba1p (
McGrath et al.,
1991). In contrast, Smt3p is activated by a heterodimer of the 636
amino-acid Uba2p, which is related to the C-terminal part of Uba1p, and the
347 amino-acid Aos1p, which is related to the N-terminal part of Uba1p
(
Johnson et al., 1997).
Similarly, the 700 amino-acid DmUba2 is related in sequence (∼40% identity
over the ∼225 amino acids of the three similarity boxes defined for other
Uba1-type and Uba2-type enzymes (
Johnson
et al., 1997;
Okuma et al.,
1999)) to the C-terminal part of the putative Drosophila
ubiquitin-activating enzyme DmUba1 (EMBL# Y15895). Therefore, we sought and
identified a Drosophila homologue of yeast AOS1 among the
ESTs from the Berkeley Drosophila Genome Project (BDGP) (see
Materials and Methods). Sequencing (Accession No. AF193554) showed that
Dmaos1 encodes a polypeptide of 337 amino acids that has 28% sequence
identity to yeast Aos1p and 40% identity to the human Aos1p homologue Sua1
(
Okuma et al., 1999), as also
observed by others (
Bhaskar et al.,
2000;
Long and Griffith,
2000). As expected, DmAos1 is related in sequence to the
N-terminal part of DmUba1 (∼37% identity over the ∼202 amino acids of
the similarity boxes as defined previously
(
Johnson et al., 1997;
Okuma et al., 1999)),
suggesting that a heterodimer of DmUba2 and DmAos1 is the Drosophila
Smt3/SUMO-activating enzyme. In support of this hypothesis, we detected an
interaction between full-length DmUba2 and full-length DmAos1 in the
two-hybrid system (
Table 3). An
attempt to use the two-hybrid system to delimit the region of DmUba2
responsible for its interaction with DmAos1 was unsuccessful
(
Table 3).

Identification of DmSmt3 and of DmSmt3-conjugated proteins

We next sought and identified a homolog of SMT3/SUMO-1 among the
BDGP EST clones (see Materials and Methods). The predicted DmSmt3 contains 90
amino acids with 48% identity to yeast Smt3p and 54% identity to human SUMO-1,
as also observed by others (
Bhaskar et al.,
2000;
Huang et al.,
1998;
Lehembre et al.,
2000). We generated polyclonal antibodies (see Materials and
Methods) and performed immunoblots on fly extracts, expecting to detect both
free DmSmt3 and DmSmt3-modified proteins. Because yeast and mammalian cells
contain Smt3p/SUMO-1-specific isopeptidases
(
Gong et al., 2000;
Kim et al., 2000;
Li and Hochstrasser, 1999;
Li and Hochstrasser, 2000),
which remove Smt3/SUMO from modified proteins, we prepared extracts both with
and without N-ethylmaleimide (NEM), an isopeptidase inhibitor. In both
extracts, the purified antibodies recognized both a polypeptide of ∼16 kDa
(presumably free DmSmt3) and many polypeptides of higher molecular weight
(presumably DmSmt3-conjugated proteins)
(
Fig. 1C). As expected, the
higher molecular-weight species were both less abundant and of lower average
molecular weight when extracts were prepared without NEM.

Interactions of DmUba2 and DmUbc9 with each other and with
DmSmt3

To test the hypothesis that DmUba2/DmAos1 and DmUbc9 are activating and
conjugating enzymes for DmSmt3, but not for ubiquitin (DmUb), we used in vitro
protein-binding assays to investigate the interactions among these proteins.
Because ubiquitin and ubiquitin-like proteins undergo proteolytic cleavage of
their C-termini to leave the sequence Gly-Gly, which is essential for both
activation and conjugation (
Kamitani et
al., 1997;
Melchior,
2000), we cloned DmSmt3 and DmUb such that they terminated with
Gly88 (DmSmt3) or Gly76 (DmUb) and were tagged with His6 at their
N-terminal ends (see Materials and Methods). We then incubated purified
His6DmSmt3 and purified His6DmUb with fly extracts,
isolated the His6-tagged proteins using Ni-NTA beads, and analyzed
the associated proteins. As expected, we found that both DmUba2 and DmUbc9
associated only with His6DmSmt3 and not with His6DmUb
(
Fig. 2A,C). The anti-DmUba2
antibodies detected not only the free form of DmUba2 (∼97 kDa) but also
species whose lower mobilities (
Fig.
2A) suggested that they might represent DmUba2 conjugated to one,
two, or three molecules of DmSmt3 (and/or some other ubiquitin-like molecule).
To ask if the interactions of DmUba2 and DmUbc9 with DmSmt3 involved
thiolester bonds, we repeated the experiments but omittedβ
-mercaptoethanol (which reduces thiolester bonds) during sample
preparation. As expected, the most abundant species now observed with the
anti-DmUba2 antibodies had an apparent molecular weight of ∼116 kDa
(
Fig. 2B), consistent with its
being DmUba2 with a single His6DmSmt3 linked by a thiolester bond.
Similarly, the anti-DmUbc9 antibodies now revealed an additional species with
an apparent molecular weight of ∼30 kDa
(
Fig. 2D), presumably
representing DmUbc9 with a single His6DmSmt3 linked by a thiolester
bond.

Conjugation of DmSmt3 to DmUba2 and DmUbc9 in vitro. Fly embryo extracts
supplemented with ATP and Mg2+ were incubated with
His6DmSmt3, His6DmUb, or no tagged protein, and proteins
were isolated using Ni-NTA beads and analyzed by SDS-PAGE and immunoblotting
(see Materials and Methods). (A,B) Immunoblotting using DmUba2-specific
antibodies. SDS-PAGE was conducted under reducing (A) or nonreducing (B)
conditions. In (A), a sample of unpurified lysate was also analyzed. The
species migrating at ∼55 kDa is presumably a breakdown product of DmUba2
(see also
Fig. 1A,B). (C,D)
Immunoblotting using DmUbc9-specific antibodies. SDS-PAGE was conducted under
reducing (C) or nonreducing (D) conditions. In C, a sample of unpurified
lysate was also analyzed. In addition to DmUbc9 (predicted molecular weight,∼
18 kDa) (
Joanisse et al.,
1998), anti-DmUbc9 antibodies also recognize a polypeptide of∼
25 kDa; as this species is evident both in the lysate and among proteins
isolated with His6DmUb, it may be a DmUb-conjugating enzyme that is
related to DmUbc9. Molecular weight markers are shown for each panel.

We also used immunoprecipitation to ask whether DmUba2 and DmUbc9 interact
in vivo. When immunoprecipitates were prepared from embryo extracts using
antibodies to either protein, the other protein was detected by immunoblotting
(
Fig. 3). Taken together, the
results of in vitro binding assays and coimmunoprecipitation suggest that
DmUba2/DmAos1 and DmUbc9 are indeed the activating and conjugating enzymes,
respectively, for DmSmt3 and that they may form a complex containing both the
E1-type and E2-type enzymes.

Coimmunoprecipitation of DmUba2 and DmUbc9. Immunoprecipitates were
prepared from embryo lysates using the antibodies indicated (see Materials and
Methods) and analyzed by immunoblotting using (A) DmUba2-specific antibodies
or (B) DmUbc9-specific antibodies. The immunoprecipitation using anti-myc
antibodies serves as a negative control. The dark band at ≥20 kDa in (B) is
presumably an allotypic form of rabbit IgG light chain in the anti-DmUbc9
antibodies that is recognized by the HRP-conjugated goat anti-rabbit-IgG.

Localization of DmUba2 during embryogenesis

To begin investigating the roles of the DmSmt3-conjugation pathway in
Drosophila, we used immunofluorescence and confocal microscopy to
characterize the intracellular localization of DmUba2. Yeast Uba2p is
concentrated in the nucleus (
Dohmen et
al., 1995), but the localization of the homologous enzyme has not
been examined in multicellular organisms. Interestingly, we observed that
DmUba2 is not exclusively nuclear during early embryogenesis. Before migration
of the nuclei to the embryo cortex after nuclear division 9, DmUba2 was found
largely in the cortex, and its distribution there appeared homogenous (data
not shown). During the interphase preceding nuclear division 10, DmUba2
gradually became organized into a cap corresponding approximately to the
cortical actin cap that forms over each nucleus
(
Fig. 4, A1-A3, C1-C3). DmUba2
was also found in the deeper cytoplasm
(
Fig. 4, B1-B3, C1-C3) and
gradually moved into the nuclei (
Fig. 4,
B2-B3, C2-C3). During mitosis, DmUba2 was dispersed in the cortex
and in the cytoplasm near the cortex (
Fig.
4, A4, B4, C4). During the three subsequent nuclear cycles, the
cap-like localization (
Fig. 4, D1-D3,
F1-F3, G1-G2, I1-I2), progressive nuclear accumulation
(
Fig. 4, E1-E3, F1-F3, H1-H2,
I1-I2), and dispersion during mitosis
(
Fig. 4, D4, E4, F4, G3, H3,
I3) of DmUba2 remained evident. However, during the successive
cycles, the cap-like cortical localization became more organized and the
degree of nuclear enrichment became more pronounced. By cycle 13, although
some DmUba2 still localized to the cortex during interphase, it was
predominantly nuclear (
Fig. 4, G1-G2,
H1-H2, and I1-I2).

Localization of DmUba2 during embryogenesis. (A-I) Localization of DmUba2
in syncytial-blastoderm embryos during nuclear cycle 10 (A-C), 11 (D-F), and
13 (G-I). Surface views of embryos in interphase (A1-A3, D1-D3, G1, and G2) or
mitosis (A4, D4, and G3) are shown; optical sections parallel to the surface
and through the middle of nuclei in interphase (B1-B3, E1-E3, H1, and H2) or
mitosis (B4, E4, and H3) and medial longitudinal optical sections of embryos
in interphase (C1-C3, F1-F3, I1, and I2) or mitosis (C4, F4, and I3) are also
shown. Each vertical set of three images (e.g., A1, B1, and C1) shows the same
embryo. Each row of interphase images (e.g., A1, A2, and A3) is ordered
according to the presumed sequence of the embryos within interphase, with the
earliest point on the left (e.g., A1), based on the assumption that DmUba2
must gradually reaccumulate in the nucleus after its dispersion during
mitosis. (J,K) Localization of DmUba2 in older embryos in stage 9 (J) or 12
(K). Medial longitudinal optical sections are shown. Arrow indicates a mitotic
domain. Bar, 10 μm (A-I); 50 μm (J and K).

Because DmUba2 was identified by its two-hybrid interaction with Sep1 and
Sep2, we examined carefully whether DmUba2 colocalized with the septins either
at the cellularization front or in cleavage furrows in mitotic domains after
gastrulation. During cellularization, most DmUba2 localized to nuclei,
accumulating preferentially at their apical ends
(
Fig. 5A,C). We did not detect
DmUba2 at the cellularization front. However, some DmUba2 remained at the
cortex, where diffuse septin staining was also observed
(
Fig. 5A,C). During mitosis in
older embryos, DmUba2 spread throughout the cell but did not become detectably
concentrated in cleavage furrows (
Fig.
5E-G, cells 1 and 2 and inset); it then moved back into the nuclei
after mitosis, with no detectable concentration at the midbody
(
Fig. 5E-G, cells 3 and 4).
Thus, we detected no substantial colocalization of DmUba2 and septins in early
embryogenesis. However, it remains possible that DmUba2 could interact with
septins at the cortex in syncytial-blastoderm embryos, during cellularization,
or in mitotic cells. DmUba2 was concentrated in nuclei of nondividing cells
and dispersed throughout the cell during mitosis throughout embryonic stages 6
to 15 (
Fig. 4J,K) (data not
shown). This was particularly striking in the CNS, where the septins, in
contrast, are enriched in axons (
Fares et
al., 1995;
Neufeld and Rubin,
1994) (J. C. Adam et al., unpublished)
(
Fig. 6D).

Localization of DmUba2 and DmSmt3 during cellularization, in mitotic
domains, and in cultured cells. Some cells are numbered for reference in the
text. (A-D) Embryos early (A,B) or late (C,D) in cellularization were stained
for Pnut (red) and either DmUba2 (green in A,C) or DmSmt3 (green in B,D).
(E-G, I-M) Mitotic domains in extended-germband embryos were double stained
for DmUba2 (E, E inset, and green in G and G inset) or DmSmt3 (K and green in
I,J,M) and either Pnut (F,L, and red in G,I,M) or tubulin (F inset and red in
G inset and J). Arrowheads in I,J indicate concentrations of Smt3 in the
regions of the chromosomes. Images of three different embryos are shown in
K-M. (H) Clone-8 cells undergoing cytokinesis were stained for DmSmt3 (green)
and Pnut (red). Bars, 10 μm (A-G, same magnification; K-M same
magnification).

Localization of DmUba2 and DmSmt3 at other developmental stages and in
other cell types. (A-C) Localization of DmUba2 to nuclei during oogenesis. Egg
chambers were double labeled with anti-DmUba2 and anti-Pnut. Arrows and
arrowheads indicate structures discussed in the text. (A) Ovariole with the
germarium on the left and successively older egg chambers moving left to
right. DmUba2 (top panel; green in bottom panel) and Pnut (middle panel; red
in bottom panel) are shown. (B) Overexposure of DmUba2 staining of an ovariole
similar to that shown in A. (C) Higher magnification of a germarium, showing
enrichment of DmUba2 (green) in nuclei of both follicle and germ cells, as
well as in the nuclei of the muscle cells in the sheath surrounding the
germarium. (D,E) Embryonic central nervous system after double staining for
Pnut (red) and either DmUba2 (green in D) or DmSmt3 (green in E). (F,G) DmSmt3
localization to nuclei of syncytial blastoderm embryos during interphase (G)
and to the chromosomes during mitosis (F). (H) Localization of DmSmt3 (green)
to interphase nuclei during embryogenesis. Embryos at stages 8 (H1), 10 (H2),
and 14 (H3) are shown.

We also examined DmUba2 localization during oogenesis. DmUba2 localized to
the nuclei of both germ cells and somatic follicle cells in the germarium
(
Fig. 6A, green arrowhead;
Fig. 6C). After encapsulation
of the germ-line cells by the follicle cells, DmUba2 remained localized to
follicle cell nuclei (
Fig. 6A,
white arrow;
Fig. 6B). DmUba2
localization to nurse cells decreased as egg chamber development progressed
(
Fig. 6A,B, green arrows), but
it remained enriched in the oocyte nucleus
(
Fig. 6A, white arrowhead). In
contrast, Pnut localizes primarily to the basal surface of the follicle cells
and is excluded from nuclei (
Fig.
6A).

Localization of DmSmt3 in embryos and cultured cells

The two-hybrid interactions between the septins and DmUba2 and DmUbc9 might
reflect a physiologically significant but transient interaction, such as might
occur if Drosophila septins, like yeast septins
(
Johnson and Blobel, 1999;
Takahashi et al., 1999), are
Smt3 modified. To explore this possibility, we used immunofluorescence to
examine DmSmt3 localization in cultured cells and in cellularizing and older
embryos. DmSmt3 did not colocalize detectably with the septins at the
cellularization front (
Fig.
5B,D). Instead, DmSmt3 localized to nuclei, with a particular
enrichment at their apical ends, as did DmUba2
(
Fig. 5A-D). In cultured cells
and in cells of post-gastrulation embryos, DmSmt3 was concentrated in nuclei
throughout interphase (
Fig. 5H,
cells 1 and 2;
Fig. 5K-M, cells
1-3). During mitosis, DmSmt3 initially appeared to spread throughout the cell
(
Fig. 5K-M, cell 5). However,
during metaphase, DmSmt3 appeared to concentrate in the region of the
chromosomes (
Fig. 5I;
Fig. 5K-M, cell 4); this was
confirmed by localizing DmSmt3 relative to the mitotic spindle
(
Fig. 5J). Strikingly, DmSmt3
was also found concentrated in a spot at cleavage furrows and midbodies both
in cultured cells (
Fig. 5H,
cells 3 and 4) and in dividing embryonic cells
(
Fig. 5K-M, cells 6-8, 10 and
13); this spot overlapped, but did not appear to coincide fully, with the
concentration of the septins in these furrows. DmSmt3 was not enriched at the
cleavage furrows early in furrow formation
(
Fig. 5K-M, cells 9, 11 and
12), but only during later stages, and it remained concentrated in the midbody
after most DmSmt3, and essentially all of the DmUba2, had reaccumulated in
nuclei (
Fig. 5H, cell 4; K-M,
cells 6-8, 10, and 13; and compare with E-G, cells 3 and 4).

We also examined DmSmt3 localization during other stages of embryogenesis.
During the syncytial cell cycles, DmSmt3 was concentrated in the nuclei during
interphase (
Fig. 6G) and
appeared to localize to the chromosomes during mitosis
(
Fig. 6F). Like DmUba2, DmSmt3
localized primarily to the nuclei of non-mitotic cells throughout the rest of
embryogenesis (
Fig. 6H),
including in the CNS (where the septins, in contrast, localized to axons)
(
Fig. 6E).

The concentration of DmSmt3 in late cleavage furrows and midbodies
suggested that one or more of the Drosophila septins might be
modified by DmSmt3. We thus tried various experimental approaches to look for
DmSmt3-modified septins. First, we used immunoblotting to look for
higher-molecular-weight forms of Pnut, Sep1 or Sep2, which might represent
Smt3-modified proteins, in extracts from embryos, adults, and cultured Clone-8
and S2 cells. Second, we immunoprecipitated Pnut, Sep1, and Sep2 from
embryonic extracts and analyzed the precipitates by immunoblotting using
anti-DmSmt3 antibodies. Third, we prepared immunoprecipitates from embryonic
extracts using anti-DmSmt3 antibodies and analyzed the precipitates by
immunoblotting using anti-Pnut, anti-Sep1, and anti-Sep2 antibodies. In each
case, we did the experiments both in the presence and the absence of the
isopeptidase inhibitor NEM. None of these approaches detected DmSmt3
modification of Pnut, Sep1, or Sep2 (data not shown). These negative results
may mean that the septins are not Smt3 modified. However, our
immunofluorescence studies detected colocalization of Smt3 with the septins
only for a brief period at the end of mitosis, and even at this stage the
overlap was not complete. Thus even if septins are Smt3 modified during this
period, they would probably comprise only a very small fraction of the total
septins in the embryo and thus might have escaped our detection.

Discussion

Possible interaction of the septins and the Smt3 conjugation
system

The Drosophila septins appear to be essential for cytokinesis in
at least some cell types, and it is likely that they have a variety of
non-cytokinesis roles as well (see Introduction). Because studies in yeast
suggest that a primary function of the septins is to serve as a matrix or
template for the organization of other proteins at the cell surface, the
identification of septin-interacting proteins should be critical to the
elucidation of septin function in Drosophila. This study began with
an attempt to identify such proteins using the yeast two-hybrid system. Of 27
positive clones identified with three septin baits, 17 contained fragments of
the septin genes themselves. Because other evidence suggests strongly that the
septins interact with each other in vivo
(
Beites et al., 1999;
De Virgilio et al., 1996;
Fares et al., 1995;
Field et al., 1996;
Frazier et al., 1998;
Hsu et al., 1998;
Longtine et al., 1996), this
result suggests that the baits used were good and that the screen was
otherwise of high fidelity. Thus, it seems likely that at least some of the
other positive clones represent genes whose products really interact with
septins in vivo.

Of the six non-septin genes identified in the screens, only two have been
investigated in detail as yet. These genes, Dmuba2 and
Dmubc9, encode the Drosophila homologues of yeast Uba2p and
Ubc9p, which catalyze the activation and conjugation, respectively, of the
ubiquitin-like molecule Smt3p (see Introduction). Several lines of evidence
suggest that the two-hybrid interactions observed between DmUba2, DmUbc9, and
the septins reflect physiologically significant interactions. First, among the
10 positive clones that did not encode septins, four contained either
Dmuba2 or Dmubc9, and Dmuba2 fragments were
isolated with two different septin baits. Second, because DmUba2 and DmUbc9
also interact with each other (see Results), the identification of both genes
independently in screens using septin baits suggests that the interactions are
relevant. Third, while our studies were in progress, it became clear that
yeast septins are extensively modified by Smt3p, although the functional
significance of that remains uncertain
(
Johnson and Blobel, 1999;
Takahashi et al., 1999;
Takahashi et al., 2001;
Johnson and Gupta, 2001) (P.
Meluh, personal communication). Fourth, several other groups also isolated
Uba2 and/or Ubc9 in two-hybrid screens using other protein baits. Several of
these interactors, including Drosophila calcium/calmodulin-dependent
kinase II (CAM-kinase II;
Long and
Griffith, 2000) and Dorsal
(
Bhaskar et al., 2000), were
subsequently shown to be Smt3 modified.

Other genetic data may also reflect an interaction between the septins and
the DmSmt3 system. The pnut septin mutation was originally identified
as an enhancer of a sina mutation that affects R7 photoreceptor
development (
Carthew et al.,
1994;
Neufeld and Rubin,
1994). Although the significance of this genetic interaction
remains unclear, it may reflect the recently discovered crosstalk between
Smt3/SUMO modification and ubiquitination. Mammalian SUMO-1 is conjugated to
the protein Mdm2, a RING-finger E3 ubiquitin ligase involved in p53
degradation (
Buschmann et al.,
2000). Modification by SUMO-1 appears to regulate Mdm2 activity
and hence the level of p53, probably by regulating the ubiquitination and
degradation of Mdm2 itself. Other RING-finger proteins, including
Drosophila Sina, interact with Ubc9 family proteins and/or are
modified by Smt3/SUMO (
Duprez et al.,
1999;
Hu et al.,
1997). Thus, it seems possible that DmSmt3 modification regulates
the activities of Sina, such as its role in downregulating the transcriptional
repressor Tramtrack (one of whose isoforms is itself DmSmt3 modified)
(
Lehembre et al., 2000;
Li et al., 1997;
Neufeld et al., 1998;
Tang et al., 1997), and that
Pnut plays a role in mediating the requisite interactions.

Finally, in several types of dividing Drosophila cells, we found
that DmSmt3 colocalizes with septins in the cleavage furrows and/or midbodies
during cytokinesis. Interestingly, DmSmt3 is not enriched in the furrow during
the early stages of furrow formation but only later, at a time when most
DmUba2 has moved back into the nuclei. Although we were unable to detect
DmSmt3 modification of Sep1, Sep2, or Pnut (despite considerable effort), it
remains possible that one or more of these proteins is modified at low levels
or that DmSmt3 is conjugated to Sep4 or Sep5. However, it is also possible
that the DmSmt3 in the midzone is conjugated to other proteins, such as the
`chromosomal passenger proteins' (
Adams et
al., 2001), which are associated with the chromosomes and then
relocate to the spindle midzone in mitosis. The involvement of such proteins
in chromosome segregation as well as in cytokinesis might help to explain the
observations suggesting that the Smt3/SUMO system is involved in chromosome
segregation (
Apionishev et al.,
2001;
Meluh and Koshland,
1995;
Tanaka et al.,
1999).

It also remains unclear whether the Drosophila septins ever serve
as a matrix/template for the localization of the DmSmt3 conjugation system.
Although our immunofluorescence studies show that DmUba2 and the septins are
sometimes in the same compartment, so that interaction would be possible, we
observed no persuasive colocalization of the proteins. Thus, elucidation of
the possible interactions between the septins and the Smt3 system in
Drosophila, and of their functional significance, will need to await
further studies using other approaches.

Conservation and possible functions of the Smt3/SUMO conjugation
pathway in Drosophila

Despite these residual uncertainties, in the course of our studies we
generated other valuable information about the Smt3/SUMO conjugation system in
Drosophila that complement and extend recent work by others on this
system. For example, our biochemical and two-hybrid studies indicate that
there are multiple DmSmt3-modified proteins, that DmSmt3-specific
isopeptidases probably exist, that DmUba2 and DmAos1 interact with each other,
and that both DmUba2 and DmUbc9 become conjugated to DmSmt3, but not to
DmUbiquitin, by thiolester bonds. While our studies were in progress, related
findings were also made by other investigators who were led by other routes to
the Smt3/SUMO system in Drosophila. In particular, several other
proteins were shown to interact with DmUba2 and DmUbc9 using the two-hybrid
system, and multiple DmSmt3-modified proteins were observed
(
Bhaskar et al., 2000;
Donaghue et al., 2001;
Joanisse et al., 1998;
Lehembre et al., 2000;
Long and Griffith, 2000;
Ohsako and Takamatsu, 1999),
supporting the hypothesis that DmSmt3 is indeed conjugated to a variety of
proteins in vivo. In addition, the DmUba2/DmAos1 interaction and the
conjugation of DmSmt3 to DmUba2 and DmUbc9 by thiolester bonds were also
observed using other methods (
Lehembre et
al., 2000;
Long and Griffith,
2000). Finally, it was shown that Dmubc9 can functionally
complement a yeast ubc9 mutation
(
Joanisse et al., 1998;
Ohsako and Takamatsu, 1999).
Taken together, these results make clear that the Smt3/SUMO conjugation system
is closely conserved between Drosophila, yeast, and mammals. Our data
also show that DmUba2 and DmUbc9 form a complex in vivo, suggesting that the
conjugation machinery may act in a concerted fashion.

The studies in other laboratories also provided clues to possible functions
of modification by DmSmt3. In particular, the semushi
(
Epps and Tanda, 1998) and
lesswright (
Apionishev et al.,
2001) mutations are both in Dmubc9, suggesting (from
their mutant phenotypes) that DmUbc9 has roles in the nuclear import of the
transcription factor Bicoid and in meiotic chromosome segregation. In
addition, two other transcriptional regulators, Dorsal and Tramtrack, as well
as CAM-kinase II, have also been shown to be modified by DmSmt3
(
Bhaskar et al., 2000;
Lehembre et al., 2000;
Long and Griffith, 2000). In
the case of Dorsal, as with Bicoid, DmSmt3 conjugation appears to promote
nuclear localization, whereas Tramtrack modification may help to regulate its
activity and/or its degradation by the proteasome, as discussed above. The
modification of CAM-kinase II may regulate its activity.

Although most suggested functions of the Smt3/SUMO system in
Drosophila, as well as in yeast and mammalian cells, center on
nuclear proteins, our immunofluorescence and two-hybrid data support the
hypothesis that there are also cytoplasmic targets. First, as discussed above,
it remains likely that in Drosophila, as in yeast, there is an
interaction between the Smt3/SUMO system and the septins, which appear to be
exclusively cytoplasmic proteins. Second, some DmSmt3-modified proteins appear
to remain both at the embryo cortex during cellularization and in the
midbodies that remain after the nuclear envelopes have reformed at the end of
cytokinesis. Third, although DmUba2 (this study) and DmUbc9
(
Joanisse et al., 1998;
Lehembre et al., 2000) are
found primarily in nuclei, considerable DmUba2 is also found in the cytoplasm
and at the cortex during the syncytial-blastoderm stage, suggesting that
DmSmt3 modification of cortical and/or cytoplasmic proteins could occur.
Finally, the cell-cycle-regulated translocation of DmUba2 between cytoplasm
and nucleus both in syncytial-blastoderm and in post-cellularization embryos
may suggest that the partitioning of the DmSmt3-conjugation system between the
cytoplasm and the nucleus is important and thus well regulated during
embryonic development.

Acknowledgements

We thank Erica Johnson and Pam Meluh for stimulating discussions; Robert
Tanguay for antibodies; Julie Brill for constructs; Tony Perdue and Susan
Whitfield for help with confocal microscopy and photography; and members of
the Pringle and Peifer laboratories for encouragement and comments on the
manuscript. This work was supported by National Institute of Health grant GM
52606 to J.R.P. and M.P. and by NIH Postdoctoral Fellowship GM19981 to
K.G.H.

De Virgilio, C., DeMarini, D. J. and Pringle, J. R.
(
1996). SPR28, a sixth member of the septin gene family
in Saccharomyces cerevisiae that is expressed specifically in
sporulating cells.
Microbiology142,
2897
-2905.

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